Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein–protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

The potent vasoconstrictor endothelin (ET) is implicated in several human disease states including hypertension, congestive heart failure, renal failure, pulmonary hypertension, ischemia, and cerebral vasospasm.1-9Two subtypes of ET receptors known as ETa and ETb have been cloned and characterized in animal and mammalian systems.10-13 A third endothelin receptor subtype has been cloned from Xenopus dermal melanophores and heart,14•15 although this subtype has not yet been described in mammalian tissues.Both ETa and ETb receptors are widely distributed in animal and human tissues.16-26 In a wide variety of animal tissues, vasoconstriction occurs via activation of ETa and/or ETb receptors depending upon the species and vascular bed under study.17-26 However, there is some controversy as to whether ETb receptors play an important role in mediating vasoconstrictor responses in mammalian tissues.20-23 Davenport et al. have reported that ETA-mediated vasoconstriction plays a major role in some human vessels, such as coronary artery, but have been unable to demonstrate ETb receptor-mediated contractions in human tissues using ETB-selective agonists such as [Ala 1,3,11,15]ET-1 and BQ 3020.20 However, Luscher et al. have reported that ETb receptor mRNA was detected by Northern blot analysis in human internal mammary artery and aortic smooth muscle cells.23 Several groups have shown that the ETb receptor agonist SRTX-6c can elicit vasoconstriction in human vessels although the magnitude of the response has been found to be considerably less than that observed for ET-1 itself.24-26 It is possible that downregulation of ETb receptors in isolated tissues is responsible for these observations.A number of peptide ET antagonists have been reported including BQ-12327•28 and FR 139317,29 which are potent ETA-selective antagonists; balanced ETa/ETb antagonists including PD 14289330 and PD 14506531 and the recently reported ETB-selective antagonist, BQ-788.32 A number of non-peptide endothelin antagonists have also been reported. These include the Shionogi steroid analog 97-13933 and several balanced ETa/ETb non-peptide antagonists, including Ro 46-2005,9 Ro 47t Department of Therapeutics.

The specific association of an SH2 domain with a phosphotyrosine (pTyr)-containing sequence of another protein precipitates a cascade of intracellular molecular interactions (signals) which effect a wide range of intracellular processes. The nonreceptor tyrosine kinase Src, which has been associated with breast cancer and osteoporosis, contains an SH2 domain. Inhibition of Src SH2-phosphoprotein interactions by small molecules will aid biological proof-of-concept studies which may lead to the development of novel therapeutic agents. Structure-based design efforts have focused on reducing the size and charge of Src SH2 ligands while increasing their ability to penetrate cells and reach the intracellular Src SH2 domain target. In this report we describe the synthesis, binding affinity, and Src SH2 cocrystal structure of a small, novel, nonpeptide, urea-containing SH2 domain ligand.

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